Signaling, transcriptional, and post-transcriptional events regulate cardiac cell fate decisions during early cardiogenesis. Disruption of such events can lead to congenital heart malformations. In particular, human mutations in transcription factors (TFs), such as GATA4, TBX5, NKX2-5 and N0TCH1, result in heart disease in children. Embryonic pathways are reactivated under stress in adult hearts, with GATA4 and MEF2C playing central roles in the transcriptional response during cardiac hypertrophy. Recent studies highlight the importance of protein-protein interactions (PPI) in dictating the transcriptional output of cardiac DNA-binding TFs. However, the complex PPIs that titrate effects of cardiac TFs have not been systematically explored. During the previous funding period of this PPG, our discoveries focused on the effects of a variety of signaling and transcriptional events that frequently culminated in combinatorial interactions between TFs and chromatin remodeling complexes to regulate cardiac gene expression. For example, manipulation of a combination of cardiac developmental TFs, including Gata4, Mef2c and Tbx5, and chromatin remodeling proteins (e.g., Baf60c), could induce the reprogramming of non-muscle cells into cardiomyocyte-like cells and promote cardiac regeneration after injury. As genome-wide TF binding data in cardiac cells accumulate, it is imperative that we understand the complex combinatorial interactions of regulatory proteins to interpret the transcriptional consequences of DNA-binding. Here, we will leverage the expertise of several of the international experts in cardiac transcription factors, a leading systems biologist, and computational biology strengths to systematically determine the complex interactomes by which the core cardiac transcriptional machinery functions to regulate gene expression during cardiac differentiation. Three discrete projects are proposed to deeply interrogate the functional consequence of selected interactomes determined by the proposed Proteomics Core. Cardiac progenitors and cardiomyocytes derived from mouse embryonic stem cells in the Cell Production Core will be used for the proteomic studies and data will be analyzed and integrated with other enhancer and chromatin data by the proposed Bioinformatics Core.